Photoremovable caging groups find extensive use in many fields ranging from cell biology to materials science. The general requirement of UV or blue light is a significant limitation due to associated toxicity and poor tissue penetration. By contrast, light between 650 and 900 nm, often referred to as the near-IR window, is cytocompatible and has significant tissue penetration (centimeters). Generally useful single photon reactions in the near-IR range would allow uncaging approaches to be applied in complex biological settings. My lab is approaching this difficult challenge by defining and then using the chemical reactions involved in the photodecomposition or photobleaching of near-IR fluorophores. These oft-encountered light-initiated processes can occur with rapid kinetics, often undesirably so. Our current efforts in this area are split in three aims. Aim 1: Development and mechanistic studies of near-IR photorelease chemistry. We have developed an uncaging reaction sequence initiated by near-IR light using readily synthesized C4'-dialkylamine-substituted heptamethine cyanines. We have shown that a variety of phenol- and amine- containing small molecules are quickly uncaged upon irradiation with low energy light. Detailed mechanistic studies involving mass spectrometry, NMR, and absorbance techniques have shown that release occurs through regioselective C-C cleavage and then hydrolysis of the C4'-amine. We are currently broadening the scope of the release process and examining aspects of the mechanism in detail using computational (collaboration with Dr. Joseph Ivanic) and experimental techniques. We have also developed an orthogonal approach to this problem that uses the a photoinduced-electron transfer process to cleave the axial ligand of silicon phthalocyanine fluorophores. Aim 2: Near-IR light control of gene expression. Several studies over the past 15 years have used UV light-mediated uncaging of small molecules in combination with inducible gene expression systems. It is quite likely that near-IR uncaging will prove beneficial to advance these techniques into increasingly complex biological settings and organismal contexts. We have shown that our approach can be used to regulate gene expression through uncaging of an estrogen-receptor antagonist in a ligand-dependent CreERT/LoxP-reporter cell line (collaboration with Dr. Susan Mackem). Our recent studies have defined chemical modifications that improve cellular uptake and retention, enabling recombination with high spatial control. We are currently applying this method in advanced models to probe pressing questions in developmental biology. Aim 3: Application of uncaging reactions for targeted drug delivery. Existing methods that use light for therapeutic interventions typically rely on the local generation of reactive oxygen species (ROS). The local delivery of potent therapeutic agents elicit alternative mechanistic paradigms, while achieving otherwise unattainable potency. We are applying our light-cleavable chemistry for targeted drug delivery (collaboration with Dr. Hisataka Kobayashi). This approach merges the unique potency of small molecule drugs with the high spatial control afforded by light release and molecular targeting. The use of tissue penetrant, cytocompatible near-IR light is critical because existing uncaging chemistries using UV or blue light would not be suitable for this application. In this area, we reported the first example of near-IR light cleavable antibody drug conjugate strategy. We have developed conjugates that release the potent anticancer natural product, duocarmycin. These conjugates can be tracked in vivo using fluorescence and uncaged attainable flux from an external CW laser source. These compounds have shown highly promising antitumor activity in in vivo models. We are currently pursuing additional optimization efforts.